The mixed-lineage leukemia gene (MLL, ALL1, HRX) encodes a 3,969 amino acid nuclear protein homologous to Drosophila trithorax and is required to maintain proper Hox gene expression. Deregulation of Hox and perhaps other gene expression causes transformation of segmental identities and contributes to human malignancy. Chromosome translocations in human leukemia disrupt MLL (11q23), generating chimeric proteins between the N-terminus of MLL and multiple translocation partners. More than 20 MLL translocation partners have been identified. They vary widely from nuclear factors to cytoplasmic structural proteins and there are no common characteristics identified among them. However, mouse models demonstrated an indispensable role played by the various fusion partners in MLL leukemias. Gene expression profiles of human leukemia bearing an MLL translocation identified a pattern of upregulated genes. Among these genes were some of the well-recognized targets of wild type MLL. This argues that the common MLL N-terminus is sufficient to confer at least some target gene specificity to MLL-fusion proteins. However, the mechanism by which MLL regulates downstream gene expression is still unclear. In preliminary studies, I demonstrate that MLL is normally cleaved at two conserved sites (D/GADD and D/GVDD) and that mutation of these sites abolishes the proteolysis. The cleavage site sequences are highly conserved from flies to mammals. MLL cleavage generates N-terminal p320 (N320) and C-terminal p180 (C180) fragments, which then interact to form a stable complex that localizes to a subnuclear compartment. Disruption of the interaction between N320 and C180 leads to a marked decrease in the level of N320 and a redistribution of C180 to a diffuse nuclear pattern. Based on these data, I propose a model in which a dynamic post-cleavage association confers stability to N320 and directs correct nuclear sublocalization of the complex, thereby controlling the availability of N320 for target gene regulation. This model predicts that MLL-fusion proteins of leukemia lose the ability to complex with C180 and instead have their stability conferred by the fusion partners, thus providing one mechanism for the altered target gene expression observed in MLL leukemic cells. Further characterization of MLL cleavage will help elucidate how MLL regulates target genes, which is crucial for both development and leukemogenesis. In this regard, I propose the following specific aims: (1) Characterize the MLL cleavage and determine its role in protein stability and nuclear sublocalization; (2) Generate knock-in mice with a noncleavable MLL; (3) Identify and characterize the protease responsible for MLL cleavage.